Development of an enhanced visual signal amplification assay for GSH detection with DNA-cleaving DNAzyme as a trigger

Abstract

Glutathione (GSH) plays a significant role in human health. Considering that dietary intake of GSH is an important way to keep GSH levels in the body, it is particularly essential to quantitate the supplemental content of GSH. In this study, an enhanced visual signal amplification assay for GSH detection in food with DNA-cleaving DNAzyme as a trigger was developed. The trigger contained a Trigger-enzyme strand (Pb2+ dependent DNAzyme sequence) and a Trigger-substrate strand. In the absence of GSH, DNAzyme was formed with Pb2+ induction and Trigger-substrate strand was cleaved to release the initiator sequence. Initiator sequence could initiate RCA reaction and generate abundant G-quadruplex sequences. After G-quadruplex/hemin DNAzyme was formed in the presence of G-quadruplex sequences, a visual signal could be produced by catalyzing ABTS. In the presence of GSH, Pb2+ would combine with GSH and there would not be following visual signal production. Thus, GSH could be detected with a “turn-off” strategy and GSH concentration was negatively correlated with color change. Besides, Nb.BbvCI nicking endonuclease was introduced to improve the visual effect. The results could be qualitatively identified by the naked eye and quantitatively analyzed by spectrophotometry (Linearity range: 10 nM to 1 µM, LOD: 3.99 nM) with good selectivity and reliability. We also developed a smartphone-based visual analysis method to make the quantitative analysis independent on a spectrophotometer (Linearity range: 10 nM to 0.5 µM, LOD: 8.17 nM). We believe that this assay showed a good potential for rapid and sensitive detection of GSH in food.